This section discusses a variety of methods for detection of compounds by the appearance of color. However, the citation of a reference or concept in this section should not be construed as an indication that the reference or concept is prior art to the present invention.
Detecting and measuring color is a convenient method for measuring the amount of a substance in solution. If the substance to be detected, i.e., the “analyte”, does not have an inherent color, a color may be produced, as surrogate for the substance, by a variety of chemical, enzymatic or immunochemical methods. This well known art is practiced, for example, in both research and clinical laboratories of biological and health care fields. The principle of colorimetry is the constant loss of light in passage through a solution by absorbance of light into the colored compound. A molecular species absorbs the same amount of light in proportion to its concentration at the same wavelength every time it is measured. Light lost as it passes through a solution is determined by the concentration of the absorbing molecules and the length of the light path. By knowing the length of the light path and the light loss and the volume of the solution, it is possible to calculate the amount, or mass, of a substance. In practice, the calculation is frequently replaced by a standard curve represented by the same reaction on a series of known amounts of the same substance that have been processed by the same reactions to produce color. In alternative methods, the amount of light lost by scattering can be determined.
Ligand Assay
The sensitivity of a calorimetric test is defined as the limit amount that may be detected reliably using the method. One way to increase sensitivity (i.e., lower the limit amount to be detected), is to use an amplifier method for producing color. Amplifiers of special interest to life science assay design are catalysts, and especially the class of biological catalysts known as enzymes. Color reactions for the detection of enzymes or for the detection of the substrates on which enzymes operate as catalysts are well known. Sensitivity may be improved further by attaching enzymes to molecules that recognize the analyte, usually referred to as ligands.
ELISA
One standard assay for detecting and quantifying an analyte in a solution is the Enzyme Linked Immunosorbent Assay, or ELISA. However, this assay can be difficult to carry out and expensive. In this assay, after the enzyme linked ligand is joined to all analyte present in the assay, the excess enzyme linked ligand that is not attached to analyte must be eliminated from the solution or it produces unwanted amplification signal. The standard assay design includes a series of steps as follows:
One example of a standard container for such assays is a 96 well plate, so called because it has a matrix of 12 by 8 wells of standard size in a standard size frame. Manufacturers may treat such plates so as to permit strong attachment of certain molecular species to the well walls.
Adding a solution containing ligand to each well on the plate at 4° C. permits attachment and retains activity of ligand. This is an “overnight” procedure.
Plates are then washed to eliminate any ligand not firmly attached to the well wall. There are three sequential washes.
Plates are most often used immediately because the ligands so attached are not stable to storage. Sample or standard analyte solutions or blanks containing reagent only are added to individual wells. The analyte species, which forms the ligand pair, attaches to the ligand on the well wall.
After some period of incubation the plates are again washed to eliminate the remaining sample solution. There are three sequential washes
Enzyme-conjugated ligand is then added to each well. Usually this ligand has specificity for another recognition site on the analyte molecule.
The plate is again washed to eliminate excess enzyme-conjugated ligand remaining in solution. There are three sequential washes. At this point the amount of enzyme attached to the wall of each well is determined by the amount of analyte also attached to the well wall.
Reagents are then added to test for the presence of the enzyme, and the color so produced relates to the amount of analyte added to each well.
Measurement is made in a photometer designed for reading the plates, called a plate reader.
It is apparent that ELISA is a tedious, time-consuming assay with many necessary steps. A manufacturer may perform the initial preparation of plates. Such manufactured plates are expensive. For example, one assay plate for performing 96 tests, may cost $650. In use, the assay using such a plate still takes approximately 5 hours to complete.
Channeling
One improvement over ELISA methods is known as “channeling” described in Gibbons et al., Methods of Enzymology 136:93. The principle of channeling is to form small, specialized particulates during the assay. The particulates permit attachment of ligands with two separate enzymes. The enzymes act in coordination, such that the product of one enzyme acts as substrate for the next. Only enzymes attached to the particles permit channeling. Enzyme not attached to particles do not produce color reaction product. Although this is a theoretical improvement over ELISA, there are considerations in formation of such specialized particles, which make this design impractical.
Other Solution Assays
Other solution assays using amplifiers are known. For example, U.S. Pat. No. 3,975,237 discloses a solution assay typically for small molecules. The assay principle is an inhibition of enzyme activity by use of a large molecule receptor, for example an antibody to the small molecular weight analyte, as competition to a small molecular version of the same analyte molecule. Methods of preparing conjugates of enzymes with analytes, and the sensitivity of assays of this nature are described. Another solution assay is described by Kricka and Ji, 1994, Clinical Chemistry 40:1828-30. This assay uses small molecule aryl boronic acids to enhance enzymatic luminescence. In a similar assay described in U.S. Pat. Nos. 5,843,666 and 5,306,621, the chemiluminescence is further enhanced by small molecule phenols. The method uses a binding partner labeled with a hydrolytic enzyme to produce a phenolic enhancer in close proximity to a peroxidase labeled specific binding partner. The mechanism of the enhancement is not known. This is a luminescence assay that uses expensive equipment that is not available to large numbers of laboratories, and has limited sensitivity.
Histochemistry
In the fields of histochemistry and cytochemistry, color contrast in tissues or cells is produced for purpose of microscopic examination or detection. Ligands that recognize tissue components and conjugated to enzymes are used as amplifiers that may then produce color with appropriate reagents. For visual examination it is possible to provide several colors of reaction product for several individual analytes with several different enzyme-conjugated ligands. This method is acceptable as long as the different analytes are located in different cells or tissue components. However, if the two analytes are present in the same location the resulting colors are additive, producing a new color which cannot be interpreted by microscopy. Another way to resolve two colors in one location is to use fluorescent markers. However fluorescence microscopy is much more expensive, and the automatic detection of fluorescence requires longer integration times, thus making automation of two color image detection impractical.
Color Photographic Development.
Exposure of color film to light produces activated silver granules in the film. Light of different colors activates silver particles in different layers of the color film. During development with a common reagent, silver grains are reduced and thereby oxidize the common developer. The oxidized developer is captured in the layer chemically combines with color couplers. It is only the product of coupling oxidized developer and color coupler that produces color. Each layer has at least one coupler producing a color reaction product specific for that layer. Scavenger molecules, sometimes called “white couplers,” prevent diffusion of developer from one layer to another. It is considered an advantage of the reaction of scavengers and colored couplers with oxidized developers if the reaction product is retained in the layer where it is formed. This is accomplished by designing or selecting couplers that are insoluble in the developer solvent both before and after coupling takes place. Some scavengers and some color couplers are designed with attachment to immobilized polymers. There are active regions on color coupler molecules and white couplers which enhance coupling to oxidized or activated photographic developers.
The chemical structure of the color couplers is highly similar to the reaction product of histochemical color producing compounds. Indeed the histochemical and cytochemical substrates are often the same as photographic color couplers with addition of a protective group on the active site. The protective group is hydrolyzed from the active region by action of the enzyme of interest. The chemical structure of photographic color developers is also very similar to substrates used in histochemistry of peroxidase reactions. The oxidized reaction product is also similar in histochemistry and in color photographic developers. In both chemical reactions the oxidation potential is also approximately the same. Use of photographic developers in detection of reaction products of hydrolytic enzymes was suggested by, for example Ornstein, 1959, Histochemistry and Cytochemistry, 7: 231 and Ornstein, 1974, Histochemistry and Cytochemistry 22:453-69, both incorporated by reference herein.
In the photographic industry it is well known that certain couplers form color more efficiently than others. By this is meant that they require less or more oxidized developer to form color. Since the amount of oxidized developer is determined by the number of sensitized silver ions, the effect is to require more sensitized silver ions for some couplers than for others. The more efficient couplers are known as 2-equivalent couplers, while the less efficient couplers are known as 4-equivalent couplers.